1. Field of the Invention
The present invention relates to a method of diagnosing allergic asthma in patients, and more particularly to a method of diagnosing allergic asthma in patients by measuring a chitin-induced response of peripheral blood immune cells obtained from the patients.
2. Description of the Related Art
To describe the related art of the present invention, definitions and characteristics of hypersensitivity, asthma and chitin have to be addressed first.
Hypersensitivity is an inflammatory reaction induced by body fluid or cell mediator from immune system, which may lead to tissue damage or even death. These reactions can be grouped into four types according to their induction mechanisms. Each type has its unique functional molecules and clinic characteristics. In clinic, most common allergic diseases including asthma, allergic rhinitis, and hay fever exhibit type I hypersensitivity.
Type I hypersensitivity is induced by allergens which might be protein or small molecule chemicals (Roitt et al., 1998). Type I hypersensitivity is a quick response with allergy reactions appearing in several minutes right after an allergen exposure. In general, these reactions have two phases—phase I in an early period of sensitization and phase II in a prolonged period after phase I. When an individual is exposed to certain allergen, his/her plasma cells will produce antibodies, especially IgEs, to fight against it. This kind of antibody has high affinity against FcεRI receptor on the surface of tissue mast cell and basophil, and low affinity against IgE receptor (FcεRII or CD23) on the surface of lymphocyte, eosinophil, platelet, and macrophage. When IgEs are bound to Fc receptors on the surfaces of mast cells or basophils, the sensitization is set out. If the individual is exposed to the same allergen again, (phase II of type I reaction), the allergen will be bound to IgE on cell receptor, causing degranulation of mast cell or basophil and release of vascular activating substance such as fast-responded histamine in huge quantity. Histamine is the major component in granules of mast cell, possessing up to 10% of granule weight. It causes smooth muscle contraction, mucus secretion increase, vessel expansion and permeability increase. Besides histamine, mast cell granule contains serotonin which causes smooth muscle contraction, increases respiration rate, facilitates histamine release and leads to overall severe allergic symptoms [Peters et al., 1998].
Asthma, one of the most common allergic diseases, is a chronic airway inflammatory disease along with major symptoms of shortness of breath, wheezing, chest tightness, and slow cough. These symptoms may last very long period of time. Most patients can recover from the disease as well as keep the disease under control to lead a normal life. Generally speaking, the cause of asthma is very complicated due to the interaction between genetic and environmental stimulation factors. For many patients, this disease might root from their infant stage. Therefore, genetic and environmental factors play important roles of establishment and development of asthma. In clinic, it is grouped as extrinsic and intrinsic asthma according to the cause of asthma. Extrinsic asthma is a type of allergic and immunogenic asthma induced by allergens from air or blood, which can be dust mite, pollen, perfume or viral antigen. Intrinsic asthma is a type of primary and non-allergic asthma caused by chronic and secondary obstructive bronchitis, which is not correlated to allergen [Kay, Trends Mol. Med. 2005; 11(4):148-152].
Chitin is the second abundant natural polymer next to cellulose. It is widely distributed in cell wall of algae and mushroom [Bulawa, 1993], and the shell of shrimp, crab and insect [Neville et al., 1976]. Chitin is a cellulose-like high molecular weight polymer. This unbranched polysaccharide polymer is formed by linking N-acetyl glucosamine and glucosamine through β-(1,4) glycosidic bond [Tharanathan and Kittur, 2003]. Due to its own highly compact crystal structure, extrinsic and intrinsic hydrogen bonds are formed in molecular level, resulting in low water solubility. Also, the sturdy hydrogen bond structure enables chitin to be physically and chemically more stable than other polysaccharide and not easy to break or distort under natural conditions [Merzendorfer and Zimoch, 2003].
Recently, due to massive supply of natural polymer, chitin is applied in all areas along with discoveries of lots of biomedical characteristics. It includes (1) non-toxicity and no obvious side effect to animal [Minami Saburo et al., 2002]; (2) bioactivity through forming micelles by absorbing food lipid in intestine to reduce micelle uptake, lower triacylglycerol and cholesterol in blood [Zacour et al., 1992], further relieve obesity, and prevent heart disease. Besides, chitin can attract negative charged chloride ion from salt and remove it out of the body for effective prevention of high blood pressure [Zacour et al., 1992]; (3) liver protection. Theoretically, chitin is believed to protect liver, inhibit tumor establishment and growth, treat stomach canker, control blood pressure, and boost immune system [Esteban et al., 2000]; and (4) high bio-compatibility, which will not induce antibody. Therefore, chitin and its derivatives are widely used in all areas, including medical, chemical engineering, agriculture, food processing and etc. [Tharanathan and Kittur, 2003; Synowiecki and A1-Khateeb, 2003; Khor and Lim, 2003].
Recent studies show that chitin can improve animal immunity, help inhibit tumor growth and protect host from pathogen infection [Murata et al., 1991 Nishimura et al., 1985], wherein 30% or 70% deacetylated chitin and carboxymethyl-chitin are the most effective material for inducing macrophage cytotoxicity to inhibit tumor growth [Nishimura et al., 1984 Nishimura et al., 1985]. Also, chitin which has been injected to guinea pig can activate C3 and C5 complement proteins therein to increase to the similar level by zymosan [Tokura et al., 1999]. Besides, other study also shows that chitin can significantly elevate macrophage secreted NO, enhancing anti-pathogen and tumor ability [Peluso et al., 1994]. Therefore chitin and chitin polysaccharide are believed to be capable of improving non-specific immune response, such as macrophage proliferation promotion, nitrogen compound secretion, and anti-bacterial and anti-tumor ability elevation.
Besides, Shibata et al. show that chitin can improve TH1 immunity against antigen Mycobacterium [Shibata et al., Infect Immune, 2001 69(10) 6123-6130]. When Mycobacterium protein (MPB-59) is used alone to immunize rat, TH2 response can be induced with the characteristics as increase of total amount of IgEs and specific IgG1s in serum. Besides, TH2 cells will produce IL-4, IL-5 and IL-10. Spleen TH1 cells produced interferon-gamma (IFNγ) and specific serum IgG2a are not detectable. If chitin is combined with MPB-50 to immune guinea pig, it shows weak TH2 response along with elevated spleen TH1 response and serum IgG2a. Therefore, chitin can improve specific immune response, promote antibody production and facilitate T cell response.
Furthermore, Shibata et al. found that chitin can significantly induce secretion of IFN-γ from TH1 cells to increase macrophage activity [Shibata et al., J Immunol, 1997 155(5) 2462-2467]. Engulfing non-allergenic chitin microparticle by macrophage leads to TH1 related cytokine production such as IL-12, IL-18 and TNF-α [Shibata et al., J Immunol, 1997 155(5) 2462-2467]. It can further activate TH1 cells or natural killer cells (NK cells) to produce IFN-γ, inhibit TH2 response, redirect the whole immune response to TH1 with effective decrease of serum IgEs and eosinophiles in lung [Shibata et al., J Immunol, 2000 164(3) 1314-1321], and finally relieve allergic symptoms. Similar results show that this type of treatment can improve airway pathological response [Musumeci et al., 2008].
Current known allergic asthma induced inflammation is highly related to allergen induced TH2 cell immune response. Therefore, chitin induced IFN-γ can be used to inhibit TH2 response in ovalbumin (OVA) sensitized asthma guinea pig and treat animal's pathological symptoms. In studying the mechanism of chitin cured ovalbumin sensitized guinea pig, it is found chitin can hardly induce sensitized or treated guinea pig immune cells to significantly produce IFN-γ, far different from the chitin-induced response of a normal guinea pig. However, this trait has not been utilized in prior art to diagnose allergic asthma in patients.